Bird brains cryosectioned and kept in cryoprotectant in a -20C freezer for ~5 years. It was preserved with acrolein and some sections were used for IHC right away. What can I do with the rest of it, besides toss it?
Can proteomics be done? Cresyl violet anatomical staining?
I would try the Cresyl Violet staining (or maybe some additional immunostaining) if that would be useful to your projects. if it doesn't work, you can throw them away and get your slide box back.
We have been able to immunostain cryosections of retina fixed with paraformaldehyde that have been stored for 5-10 years at -20 to -30 degrees C. This does not work for all antigens, autofluorescence tends to increase, and the tissue quality tends to decrease a bit with time, so the results usually are not optimal but can still be useful. I would expect that nissl staining quality should roughly parallel the immunostaining. I have not worke with acrolein fixed tissue, so I can't comment specifically on how that would compare to paraformaldehyde fixed tissue.
I am absolutely convinced that the decline in antigenicity and quality of frozen tissue sections is accelerated by repeated freeze-thaw cycles (like when people pull the slide box out of the freezer and take forever to select slides to stain).If the slide boxes have been at the back of the freezer undisturbed for a long time they may have a better chance of wroking than slides that sat at the front of the freezer the whole time.
In comparison, we have found that the quality and antigenicity of frozen sections from unfixed mouse brain declines much more rapidly and severely and last only about 3 months maximum under those conditions. Nissl and myelin stain quality decreased in parallel with immunostaining. We also have done some nissl and myelin staining on these unfixed tissue sections.
I think this material might be okay for some stainings, but for proteomics this is not optimal.
Thank you both for your input! It sounds like there is just too much uncertainty to make it worth while to stain for anything. The quality is likely far below most tissues kept in a lab setting. The sections have been floating in liquid cryoprotectant and exposed to varying temperatures over the years. The steps needed to validate any staining would be out of my reach at the moment too since I don't have freshly preserved tissues. They were left over from my dissertation experiments. My options were to junk them before I left or take them with me and hope for the best. I've carried the weight long enough, I guess! I just wanted to hear from others with more experience in this area that there was NOT a sure thing I was walking away from. Thank you for taking the time to respond.
In our lab, we have successfully immunohistochemically stained formaldehyde (4%) fixed brain tissue stored at -20°C in cryoprotectant for 3-5 years. As previously mentioned, this may not work for all antigens. We also get good results with histological staining (HE, methylene blue…).
It is definitely worth trying to perform immunohistochemical or histological staining before tossing the material.
On the other hand, we have found that frozen brain sections (vacuum-packed and stored in a freezer at -20 or –80 °C) are useful for immunostaining as well as western immunoblotting or in situ hybridization, for longer periods (up to 10 years).
Hi Larisa, you've restored my hope that there may be a use for the tissues after all. I have to at least try if there is a chance. Now I have to find someone with the lab setup and supplies ready to go. Thanks for sharing what you know!
It may depend on the cryoprotectant used (and the reliability of the freezer), but I have successfully stained sections of mouse brain stored for 15 years in 50% glycerol/PBS/0.1% PVP40 at -20C.
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